Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 33470-153 FK
- Expiration date of the lot/batch: 30 Apr 2009.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: The sponsor guarantees the storage stability at least until 30 Apr 2009.
- Solubility and stability of the test substance in the solvent/vehicle: Because the test substance is applied undiluted or minimally moistened with doubly distilled
water, no analysis of the test substance in a vehicle is required.

FORM AS APPLIED IN THE TEST (if different from that of starting material): minimally moistened with doubly distilled water.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Skin model: Epi-200, Supplier: MatTek Corp., Ashland MA, USA
Details on animal used as source of test system:
SOURCE ANIMAL
- Source: human
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epiderm model (Epi-200)
- Tissue batch number(s): Lot No. Kit 9450
- Date of initiation of testing: 18.07.2007

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
- Observable damage in the tissue due to washing: Skin more or less detached from supporting diaphragm in all reaction chambers before washing. Washing resulted in full detatchment and ripping skin in pieces.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ mL assay medium.
- Incubation time: 3 hours.
- Spectrophotometer: Image Reader Spectra SL T.
- Wavelength: 570 nm.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: killed tissue.
- Procedure used to prepare the killed tissues (if applicable): killed by twice freezing at -20°C and thawing at room temperature.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL
Duration of treatment / exposure:
3 min, 1 hour
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1, mean of 2 replicate tissues/ 3 min exposure period
Value:
105
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1, mean of 2 replicate tissues/ 1 hour exposure period
Value:
112
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: Skin more or less detached from supporting diaphragm in all reaction chambers before washing. Washing resulted in full detatchment and ripping of skin in pieces. However, validity of the study was indicated by fullfillment of acceptance criteria.
- Direct-MTT reduction: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Any other information on results incl. tables

Table 1: Individual and mean OD570 values, individual and mean viability values after 3 min exposure.

   OD570
tissue 1
 OD570
tissue 2
mean
OD570
viability
[% of NC]
NC 1.759 1.583 1.671 100
Test material 1.796 1.709 1.752 105
PC 0.263 0.203 0.233 14

NC: negative control

PC: positive control

Table 2: Individual and mean OD570 values, individual and mean viability values after 1 hour exposure.

   OD570
tissue 1
 OD570
tissue 2
mean
OD570
viability
[% of NC]
NC 1.641 1.683 1.662 100
Test material 1.779 1.933 1.856 112
PC 0.223 0.293 0.258 15

NC: negative control

PC: positive control

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test material showed no skin corrosion potential in the EpiDerm™ in vitro Corrosivity-Test under the test conditions chosen.
Executive summary:

The potential of the test substance to cause dermal corrosion was assessed by a single topical application of 10 mg of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).

Two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability.

The following results were obtained in the EpiDerm™ Corrosivity-Test:

The test substance is not able to directly reduce MTT.

The mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 105 % and it was 112 % after an exposure period of 1 hour.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test material shows no skin corrosion potential in the EpiDerm™ in vitro Corrosivity-Test under the test conditions chosen.